Oxidative DNA damage, and exocyclic DNA adducts are involved in the initiation of mutations that lead to human cancer. This Program Project focuses on characterizing the mechanisms by which damaged DNA is recognized, replicated and repaired. The program theme involves elucidation of relations among molecular structure, energetics and biological function, incorporating the expertise of chemists, biophysicists, structural, cell and molecular biologists in this endeavor. As a result of these efforts, novel biological systems have been developed that reflect the mutagenic specificity of a single DNA adduct. X-ray crystallography and NMR spectroscopy are used to establish structures of key enzymes complexed to oxidatively damaged DNA. Significant correlations between structure, energetics, and biological activity are expected to emerge from this interdisciplinary research. We will characterize conformational changes and energetic perturbations induced by DNA damage, and assess how these perturbations modulate damage recognition, DNA repair, and miscoding events. We will establish three-dimensional structures and characterize the energetics of DNA glycosylases and DNA polymerases bound to their cognate DNA substrates. The molecular and energetic origins of the functions of these key enzymes, as defined by structural studies and thermodynamic measurements, will be explored by site-directed mutagenesis techniques, thereby illuminating molecular mechanisms of mutagenesis and carcinogenesis. The long-term goals of the Program include: (i) elucidating mechanisms of translesion synthesis, mutagenesis, and base excision repair in human cells;(ii) relating the molecular structure of DNA polymerases and glycosylases to their biological functions of replication and repair of damaged DNA;and (iii) characterizing the structural and energetic consequences of DNA lesions, relating lesion-induced alterations to biological function as manifest in DNA recognition, repair, and replication by DNA glycosylases and polymerases.